This application is a continuation-in-part of commonly assigned applications U.S. Ser. No. 190,271, filed May 4, 1988, PCT/US89/01950, filed May 4, 1989, and PCT/US89/01951, filed May 4, 1989. The subject matter of said applications is incorporated by reference.
This invention relates generally to methods for enhancing the rate of cleavage or formation of peptide bonds. More particularly, this invention relates to methods for enhancing the rate of cleavage or formation of specific metastable peptide bonds within protein or peptide molecules by contacting such molecules with a rate-enhancing antibody.
Several publications are referenced in this application by Arabic numerals within parenthesis. Full citation for these references are found at the end of the specification immediately preceding the claims. The references more fully describe the state of the art to which this invention pertains as well as certain aspects of the invention itself.
It is known that certain peptide sequences in proteins are metastable. These sequences, also referred to as xe2x80x9csensitopes,xe2x80x9d are susceptible to spontaneous chemical reactions such as deamidation, isomerization, racemization, and in some cases peptide bond cleavage. As such, they may be target sites for antibodies that enhance the rate of chemical reactions that occur at such sensitive sites.
It is known that certain synthetic peptide sequences are particularly susceptible to spontaneous reactions (1). Asparagine, aspartic acid, glutamine and glutamic acid are amino acid residues that are frequently associated with susceptible sequences, and it has been proposed that the amino acid residues flanking these particular side chains can determine the particular susceptibility of these sites within peptides (2). It has also been observed that the structural features around these sites in intact proteins can also influence the stability of these sites to spontaneous chemical modification (3).
More specifically, it is known that polypeptides containing the dipeptide sequences, ASN-PRO, ASN-GLY, ASP-PRO, ASP-GLY GLN-X or GLU-X, wherein X is any amino acid, undergo hydrolysis at a much higher rate than other dipeptides. This instability is due to the formation of a cyclic structure resulting from intramolecular attack of the side chain amide or acid on the peptide bond between the two amino acids. However, these metastable bonds are reported to be more stable in native (not denatured) proteins (2).
Antibodies have previously been generated that catalyze acyl transfer reactions (4), sigmatropic rearrangements (5), intramolecular cyclization (6), and peptide bond hydrolysis (7). It has been speculated that such antibodies may be particularly suited to performing substrate assisted catalysisxe2x80x94i.e., catalyzing the reaction of a substrate containing a reactive nucleophile or catalyst within the molecule undergoing transformation.
It is known that antibodies raised against peptides are able to bind to the same sequence when the latter are located within an intact protein. For example, antibodies elicited against a peptide comprising amino acids 1-15 of tumor necrosis factor (TNF) are able to bind to native tumor necrosis factor and in doing so, inhibit its interaction with a cell surface receptor (8). Similarly, antibodies against a peptide comprising amino acids of the gp 120 coat protein from HIV cross-react with the intact virus and inhibit the interaction of the virus with its cellular receptor, CD4 (9). In another example, monoclonal antibodies raised against a peptide comprising amino acids 67-83 of hen egg lysozyme were able to cross-react with the intact protein and are able to recognize other avian species of lysozyme whose sequences within the epitope are substantially similar (10).
While methods for preparing catalytic antibodies have been described, and while methods for binding noncatalytic and catalytic antibodies to antigens or substrates of interest have been described, the art has heretofore not provided methods of enhancing the rate of cleavage or formation of certain metastable peptide bonds known to undergo spontaneous hydrolysis.
It is a primary object of this invention to provide methods for enhancing the rate of cleavage or formation of metastable peptide bonds within protein or peptide molecules.
It is a further object of the invention to provide methods for enhancing the rate of cleavage or formation of metastable peptide bonds, e.g., ASN-PRO, ASN-GLY, ASP-PRO, ASP-GLY, GLN-X or GLU-X, wherein X is any amino acid, by contacting the peptide or protein molecule containing the metastable peptide bond with a rate-enhancing antibody which is prepared by a rational design method according to the invention.
It is still a further and related object of the invention to provide methods for enhancing the rate of hydrolysis of specific peptide bonds in protein or peptide molecules by contacting such molecules with a rate enhancing antibody which promotes the natural tendency of these bonds to form a cyclic intermediate structure by intramolecular attack of the amide or acid group of the aspartic or glutamic acid or asparagine or glutamine side chains on the peptide bond.
These and other objects of the invention are achieved in an antigen for elicitation of a rate-enhancing antibody, said antigen containing a hapten having a metastable bond.
One embodiment of the invention is an antigen for elicitation of an antibody capable of enhancing the rate of reaction of a substrate of interest at the site of a metastable bond, said antigen containing a hapten which mimics said substrate of interest at or near the said site of said metastable bond.
A further embodiment of the invention is an antibody which enhances the rate of modification of a metastable bond in a substrate of interest, said antibody having been prepared by a process comprising the steps of: selecting the specific metastable bond to be modified; selecting an antigen comprising a hapten which mimics said substrate at or near the said site of said metastable bond; exposing cells capable of producing antibodies to said antigen and thereby generating antibody producing cells; hybridizing said antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening said plurality of monoclonal antibodies to identify a monoclonal antibody which binds to an epitope at or near the metastable bond to be modified and enhances the rate of modification of said metastable bond.
A further embodiment of the invention is a method for preparing antibodies which enhance the rate of cleavage or formation of a metastable bond of interest comprising the steps of: selecting the specific metastable bond to be cleaved or formed in a protein or peptide molecule substrate of interest; selecting an antigen comprising a hapten which mimics said substrate at or near the said site of said metastable bond; exposing cells capable of producing antibodies to said antigen and thereby generating antibody producing cells; hybridizing said antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening said plurality of monoclonal antibodies to identify a monoclonal antibody which binds to an epitope at or near the metastable bond to be modified so as to enhance the rate of modification of said metastable bond.
A yet further embodiment of the invention is a method for enhancing the rate of modification of a specific metastable bond within a protein or peptide molecule substrate of interest which comprises contacting said substrate with an antibody under conditions sufficient for said antibody to bind to said substrate at an epitope at or near said specific metastable bond and to enhance the rate of reaction.
A still further embodiment of the invention is a method for enhancing the rate of modification of a specific metastable bond within a protein or peptide molecule substrate of interest which comprises contacting said substrate with an effective amount of an antibody, under conditions sufficient for said antibody to bind to said substrate at an epitope at or near said specific metastable bond, and thereby enhance the rate of said reaction, said antibody having been produced by the method of: selecting the specific metastable bond to be modified; selecting an antigen comprising a hapten which mimics said substrate at or near the said site of said metastable bond; exposing cells capable of producing antibodies to said antigen and thereby generating antibody producing cells; hybridizing said antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening said plurality of monoclonal antibodies to identify a monoclonal antibody which binds to an epitope at or near the metastable bond to be modified.
The invention, as well as other objects, features and advantages thereof will be understood more clearly and fully from the following detailed description.
The invention embodies an antigen wherein the metastable bond is selected from the group consisting of ASN-X, ASP-X, GLN-X, GLU-X, LYS-X, and HIS-Y-X, where X and Y are any amino acid.
In particular, the invention embodies an antigen containing a hapten which is immunologically cross reactive to an amino acid sequence at or near the said site of said metastable bond.
More in particular, the invention embodies an antigen wherein said hapten is comprised, of an amino acid sequence of at least two amino acids.
Still more in particular, the invention embodies an antibody elicited by said antigens wherein the said metastable bond is selected from the group consisting of ASN-X, ASP-X, GLN-X, GLU-X, LYS-X, AND HIS-Y-X, wherein X and Y are any amino acid and, wherein the identity of the said metastable bond is determined by subjecting the substrate of interest to modification under art-known conditions and analyzing the products obtained in such modification.
Definition of Terms
In its broadest sense, the term xe2x80x9cantigenxe2x80x9d is defined as a molecule which induces the formation of an antibody. As used herein, the term xe2x80x9cantigenxe2x80x9d means a molecule which is inherently immunogenic, a hapten according to the invention or an immunogen which comprises a hapten according to the invention coupled to a carrier molecule by a suitable coupling moiety. Carrier molecules include, for example, keyhole limpet hemocyanin (KLH), thyroglobulin, chicken immunoglobulin, ovalbumin, bovine serum albumin (BSA), T-helper peptides, etc. xe2x80x9cCoupling moietiesxe2x80x9d as used herein refer to biotechnological cross-linking reagents well known in the art (e.g., commercially available from Pierce, Rockford, Ill.) and include, for example, Traut""s reagent, dissuccinyl suberate, etc.
The term xe2x80x9cantibodyxe2x80x9d includes whole immunoglobulins and fragments thereof which contain the binding site for the antigen.
The term xe2x80x9crate enhancing antibodyxe2x80x9d refers to antibodies according to the invention which recognize and bind to epitopes on proteins or peptide molecules containing a metastable peptide bond and thereby stoichiometrically or catalytically (as these terms are defined below) enhance the rate of the reaction.
The term xe2x80x9cmetastable peptide bondxe2x80x9d includes all bonds which have a propensity for undergoing spontaneous reactions of formation or cleavage. The term xe2x80x9cspontaneous reactionxe2x80x9d refers to a reaction at a specific position within a peptide sequence which proceeds at a rate higher than usually observed for peptide bonds. In particular, ASN-PRO, ASN-GLY, ASP-PRO, ASP-GLY, GLN-X or GLU-X, wherein X is any amino acid, are known to undergo a spontaneous peptide bond hydrolysis mediated by formation of a cyclic intermediate.
The term xe2x80x9cdipeptide analogxe2x80x9d as used herein refers to a structure in which the normal amide bond (i.e., xe2x80x94COxe2x80x94NHxe2x80x94) between the two amino acids has been replaced by an array of atoms as defined above. Additional amino acid residues may be incorporated to surround the dipeptide analog to form a polypeptide. The moieties surrounding the dipeptide analog contain peptide bond linkages which can be altered such that the naturally occurring Cxe2x95x90O group is replaced by NH, O, S, CH2, CF2 or Cxe2x95x90S and/or the naturally occurring NH group is replaced by O, S, CH2, CF2, Cxe2x95x90O or Cxe2x95x90S. For example, the moieties can be retropeptides in which the Cxe2x95x90O and NH groups of the amide bonds are interchanged.
The term xe2x80x9chaptenxe2x80x9d as used herein is defined as a molecule which can act as an epitope. Haptens may contain an amino acid sequence of at least two amino acids which are identical to or mimic the region of a peptide or protein containing the metastable bond of interest. A hapten may also comprise an analog such as a dipeptide analog as heretofore defined.
The term xe2x80x9cnaturally occurring amino acidxe2x80x9d as used herein includes the twenty essential alpha-amino acids and other alpha-amino acids which may or may not be found in proteins. These amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 4-hydroxyproline, 5-hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, beta-alanine, gamma-aminobutyric acid, homocysteine, homoserine, citrulline, ornithine, canavanine, djenkolic acid and beta-cyanoalanine. An amino acid consists of a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom and a distinctive group referred to as a xe2x80x9cside chain.xe2x80x9d The term xe2x80x9canalog of said side chainxe2x80x9d as used herein is defined as a side chain of a naturally occurring amino acid in which one or more moieties of the naturally occurring side chain is replaced by one or more different moieties which substantially corresponds to the naturally occurring moiety. Those side chains containing a hydroxy group can be glycosylated, phosphorylated, sulphonylated or protected by a hydroxy protecting group. The hydroxy group of any of the side chains may be protected by any number of suitable hydroxy protecting groups well known in the art. These include, for example, a tertiary butyl ether group.
Binding of an antibody xe2x80x9cat or near the site of interestxe2x80x9d refers to binding of an antibody directly to the metastable bond of interest, binding to peptide sequences adjacent to the metastable bond of interest, or to binding both directly on the metastable bond of interest and on the amino acid sequences on one side or both sides of the metastable bond of interest.
A xe2x80x9ccatalyticxe2x80x9d antibody is an antibody which is capable of changing the rate of a chemical reaction, all other conditions (e.g., temperature, reactant/substrate concentration, etc.) being the same, and which is not consumed in the reaction, and which has the capability of converting multiple moles of reactant/substrate per mole of catalytic antibody. From a mechanistic viewpoint, it binds the reactant/substrate, effects the accelerated conversion of the reactant/substrate to the product and then releases the product, changing the rate of the chemical reaction without shifting the position of the equilibrium. The aforementioned definitions are characteristics of ideal catalysts. However, in practice, even the best of catalysts become poisoned or deactivated by contamination in the reaction system or as a result of chemical or physical destruction during the reaction process. For reasons well known in the art, the true operation of a catalyst may be obscured by components of the reaction system or by the condition of the reaction environment. Rate enhancing antibodies directed to metastable peptide bonds may be described as catalytic antibodies if the antibody is released from the epitope after the reaction is complete.
A xe2x80x9cstoichiometricxe2x80x9d antibody is an antibody which enhances the rate of the chemical reaction stoichiometrically, i.e., it enhances the rate of the reaction, but unlike a catalytic antibody, is stoichiometrically consumed during the reaction. Rate enhancing antibodies directed to metastable peptide bonds may be described as stoichiometric antibodies if the antibody remains bound to the epitope after the reaction is complete or is altered by the reaction, and thus cannot promote additional reactions.
Metastable bonds are the preferred modification sites for the methods of the invention. For example, sequences containing the following amino acid combinations, ASN-GLY, ASN-PRO, ASP-GLY, ASP-PRO, GLN-X, or GLU-X, wherein X is any amino acid, are known to be metastable in denatured proteins or small peptides and to undergo spontaneous hydrolysis. When present in a native protein, these bonds are more stable, but the binding of an antibody to an epitope at or in proximity to the metastable bond destabilizes the bond and enhanced rate of cleavage is obtained.
The identity and location of a metastable bond within a protein or peptide molecule of interest may be known or may be established by reference to various methods available to the art (11,12). Such information may be available in various forms and with various levels of precision and may include the three-dimensional structure of the protein or peptide molecule, computer models or predicted structures thereof, or hydrophilicity profiles.
An empirical method for identifying suitable metastable bonds includes subjecting the protein or peptide molecule of interest to art-recognized modification conditions for a time sufficient to permit modification to occur. One skilled in the art will appreciate that other methods can be used to induce autolysis, such as, for example, incubation of the protein in EDTA (ethylene diamine tetracetic acid) at varying temperatures (14-16), hydroxylamine (17) or dilute acids (18, 19), as well as varying the temperature.
Thereafter, the modified fractions can be identified and the metastable bonds at which modification has occurred can be identified.
Protein or peptide molecules which may advantageously be modified according to the methods of the invention include immunoglobulin E (hydrolysis), tumor necrosis factor (hydrolysis), and human immune deficiency virus (hydrolysis).
Once the metastable bond to be modified has been identified by the method described above, an antigen can be obtained or synthesized for use in an immunological method for eliciting antibodies. The antigens are desirably small peptides or analogs thereof which contain the metastable bond of interest or an analog of that metastable bond of interest. The antigen then can be employed as an immunogen to elicit through either in vitro or in vivo techniques antibodies having the desired rate-enhancing properties.
Broadly, the method comprises exposing cells capable of producing antibodies to the immunogen and thereby generating antibody producing cells; hybridizing the antibody producing cells with myeloma cells and thereby producing a plurality of hybridoma cells each producing monoclonal antibodies; and screening the plurality of monoclonal antibodies to identify a monoclonal antibody which catalyzes the chemical reaction of interest. The monoclonal antibody so identified may then be replicated, again by either in vivo or in vitro techniques, to obtain a quantity sufficient to catalyze the chemical reaction of interest.
The preferred immunogens of the invention comprise peptides having metastable sites or amino acid side chains that can participate in the catalytic process by a substrate assisting mechanism. These include sequences comprising: ASN-X; ASP-X; GLN-X; GLU-X; LYS-X; and HIS-Y-X wherein Y and X are any amino acids. Other immunogens as may be found by these empirical hydrolysis-fragment analysis techniques of the invention may also be used. Immunogens comprising cyclic analogs designed to induce the peptide or protein substrate to undergo intramolecular catalysis by creating an antibody combining pocket complementary to a reaction pathway of the specific reaction to be catalyzed may also be used.
The detection of antibodies with the desired activity and specificity is achieved by screening the hybridomas once they have been elicited. For example, screening may be achieved by high performance liquid chromatography (HPLC) or spectrophotometric methods (ELISA). Monoclonal antibodies are elicited in vivo by modification of the technique disclosed by Koprowski et al. in U.S. Pat. No. 4,196,265, issued Apr. 1, 1980, which is hereby incorporated by reference. The details of that process are known in the art. A series of monoclonal antibodies directed to a specific antigen are prepared under suitable conditions. This involves first immunizing BALB/C mice with an appropriate antigen. The antigen comprises a hapten according to the invention bound to a peptide or other carrier molecule.
Antibody-producing lymphocytes are then removed from the spleens of the immunized mice and hybridized with myeloma cells such as SP2/0 cells to produce hybridoma cells. These hybridoma cells are then plated in the wells of microtiter plates. The series of monoclonal antibodies being produced by the hybridoma cells is screened under appropriate conditions to identify monoclonal antibodies which catalyze the desired reaction under appropriate conditions. Alternatively, the medium may be tested for antibodies that bind to the immunogen and the hybridomas producing these antibodies then expanded in tissue culture or grown in vivo. Screening may be conveniently accomplished by treating a standardized solution of the reactant with an aliquot of medium withdrawn from a microtiter well and measuring the presence of the desired product by conventional instrumental methods. This measurement may be readily conducted, for example by spectrophotometric methods or by gas-liquid or high pressure liquid chromatography. By comparison with standardized samples of the desired product or reactant, rates of reaction may be quantified. In this manner, wells containing hybridoma cells producing monoclonal antibodies are identified. The selected hybridoma cells are then cultured to yield colonies.
These colonies may be further propagated in vitro or in vivo systems. In the latter case, mice such as syngeneic BALB/C mice are inoculated intraperitoneally with the selected hybridoma cells and produce tumors, generally within two or three weeks. These tumors are accompanied by the production of ascites fluid which contains the desired monoclonal antibodies. The monoclonal antibodies are then separately recovered from the ascites fluid by conventional methods such as ultrafiltration, ultracentrifugation, dialysis and immunoaffinity chromatography.
Antibodies elicited with the immunogens of the invention are xe2x80x9csite specificxe2x80x9d in that they are designed only to catalyze modification of the metastable bond of interest. Likewise, these antibodies are designed only to catalyze the formation of bonds from the termini of moieties having certain structural conformations at those termini. Rationally designed immunogens according to the invention may be used to elicit a site specific antibody capable of cleaving bonds at specific sites in a protein or peptide molecule to produce two or more cleavage products or to catalyze the formation of bonds wherein those cleavage products having the right structural conformation are joined.